Setting Ten community and tertiary care hospitals in the United States.

Patients Between November 2001 and March 2003, 123 patients aged 38 to 82 years
consented, 57 were randomly assigned, and 47 completed the protocol.

Interventions In a ratio of 1:2:2, patients received 5 weekly infusions of placebo
or ETC-216 at 15 mg/kg or 45 mg/kg. Intravascular ultrasound was performed
within 2 weeks following ACS and repeated after 5 weekly treatments.

Main Outcome Measures The primary efficacy parameter was the change in percent atheroma volume
(follow-up minus baseline) in the combined ETC-216 cohort. Prespecified secondary
efficacy measures included the change in total atheroma volume and average
maximal atheroma thickness.

Conclusions A recombinant ApoA-I Milano/phospholipid complex (ETC-216) administered
intravenously for 5 doses at weekly intervals produced significant regression
of coronary atherosclerosis as measured by IVUS. Although promising, these
results require confirmation in larger clinical trials with morbidity and
mortality end points.

Figures in this Article

In a small village in northern Italy called Limone sul Garda live approximately
40 carriers with a naturally occurring variant of apolipoprotein A-I known
as ApoA-I Milano. Individuals with ApoA-I Milano
are characterized by very low levels of high-density lipoprotein cholesterol
(HDL-C) (10-30 mg/dL [0.25-0.78 mmol/L]), apparent longevity,1 and
much less atherosclerosis than expected for their HDL-C levels.2 The
ApoA-I Milano protein differs from native ApoA-I in that cysteine is substituted
at position 173 for arginine allowing disulfide-linked dimer formation. Recombinant
ApoA-I Milano has been formulated in a complex with a naturally occurring
phospholipid to mimic the properties of nascent HDL (ETC-216, Esperion Therapeutics,
Ann Arbor, Mich). Studies in mice and rabbits with experimental atherosclerosis
have demonstrated that rApoA-I Milano/phospholipid complexes rapidly mobilize
cholesterol and thereby reduce atherosclerotic plaque burden. The antiatherosclerotic
effects (reductions in plaque lipid and macrophage content) occur in animals
as rapidly as 48 hours after a single infusion.3

We hypothesized that short-term weekly infusions of ETC-216 might rapidly
regress coronary atherosclerosis in patients following an acute coronary syndrome
(ACS). To test this hypothesis, we conducted a prospective, randomized, double
blind, placebo-controlled clinical trial of ETC-216 using intravascular ultrasound
(IVUS) to measure atheroma burden. Intravascular ultrasound is an imaging
modality that provides detailed images of the vessel wall using a using a
high-frequency (40 MHz) miniaturized transducer.4 A
motorized pullback device is used to generate cross-sectional images throughout
the length of the vessel, enabling precise quantification of atherosclerotic
disease burden. This approach has been used recently in studies designed to
assess the effect of pharmacological agents on atherosclerosis.5- 7 We
used IVUS to measure change in atheroma volume after a regimen consisting
of 5 infusions of ETC-216 or placebo at weekly intervals.

METHODS

Study Protocol

The ApoA-I Milano Trial was a randomized, double-blind, multicenter,
parallel-treatment study to assess the effects of 2 different doses of ETC-216
or placebo administered weekly for 5 weeks on coronary atheroma volume as
measured by IVUS in patients with ACS. The study was conducted between November
2001 and March 2003. The institutional review boards of all participating
centers approved the protocol and written informed consent was obtained from
patients prior to any study-related procedures.

Inclusion Criteria

Patients aged 30 to 75 years who required diagnostic coronary angiography
for clinical indications within 14 days after an ACS, defined as unstable
angina, non–ST myocardial infarction, or ST-elevation myocardial infarction,
were eligible to be considered for the study. Angiographic inclusion criteria
required the presence of an obstructive lesion in a major epicardial vessel
with at least a 20% luminal diameter narrowing by visual (angiographic) estimation.
The initial IVUS examination was performed in a single coronary artery within
2 weeks of the ACS event. Patients were required to have a target vessel for
IVUS interrogation with no more than 50% luminal narrowing throughout a segment
with a minimum length of 30 mm (target segment). The target vessel must not
have undergone previous angioplasty nor have been a candidate for intervention
at the time of baseline catheterization.

The protocol specified that patients receive the customary standard
of care for ACS. A core laboratory at the Cleveland Clinic Foundation screened
the initial IVUS examination, and the patient was randomly assigned only if
the ultrasound study met prespecified image-quality requirements.

Randomization and Allocation Concealment

Patients were randomized to 3 treatment groups in a 1:2:2 ratio—placebo
or a low (15 mg/kg) or a high dose (45 mg/kg) of ETC-216. A consulting statistician
using SAS version 8.02 (SAS Inc, Cary, NC) generated the randomization sequence
prior to the start of the study. A list of randomization assignments for each
center was placed in a sealed envelope and sent directly to each center's
pharmacist. Patients were enrolled sequentially as they became qualified.
Blocks of 5 patients (2:2:1) comprised the code. The pharmacist at each site
was unblinded and was aware of allocation prior to randomization but had no
other role in the conduct of the study.

Intervention and Blinding

The ETC-216 treatment was prepared as a sterile injectable solution
of a recombinant human apolipoprotein A-I Milano/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine complex prepared in a 6-µL
phosphate buffer at pH 7.4, 6.4% sucrose and 0.9% mannitol. The ETC-216 treatment
was supplied frozen by Esperion Therapeutics Inc, Ann Arbor, Mich, in vials
containing protein and phospholipid concentrations in a ratio of 1:1 at approximately
15 mg/mL. The dose calculations were based on the protein content of the dosing
solution. The placebo consisted of the 0.9% normal saline. Both active drug
and placebo were shrouded in an identical sealed opaque cover. Pharmacists
were instructed to deliver the infusate to the study physicians and nurses
without revealing the treatment assigned. All other study personnel and the
patients had no access to randomization assignments.

The study drug was administered as an intravenous infusion at weekly
intervals for 5 doses. Within 2 weeks of the final dose, patients underwent
repeat IVUS of the originally imaged coronary vessel to permit comparison
of paired examinations at the 2 time points.

Patients were permitted to continue clinically indicated medications
for dyslipidemia as long as their doses remained stable for the study duration.
Patients in whom lipid-lowering therapy had been initiated or discontinued
during the 6 weeks before screening were excluded.

Outcome Measures

The primary outcome measure was change in percent atheroma volume (follow-up
minus baseline) as measured by IVUS in both ETC-216 groups combined. Prespecified
secondary outcome measures were average change in total atheroma volume, change
in average maximal atheroma thickness, and change in atheroma volume in the
most severely and least severely diseased 10-mm-long subsegments. Quantitative
angiographic change and adverse events were also assessed.

IVUS and Angiography

Following diagnostic coronary angiography, the operator selected a single
major epicardial vessel for interrogation based on the criteria previously
noted. If more than 1 vessel met all inclusion criteria, the investigator
was instructed to select the vessel with the longest and least angulated segment
suitable for an IVUS pullback. Briefly, after anticoagulation with heparin
and administration of 100 µg to 300 µg of intracoronary nitroglycerin,
a 0.014-inch guidewire was subselectively placed in the vessel selected for
interrogation. A 40-MHz, 2.6 F (0.87 mm) IVUS catheter (Atlantis, Boston Scientific
Scimed, Inc, Maple Grove, Minn) was advanced into the target vessel and the
transducer positioned just distal to a side branch (distal fiduciary site).
The IVUS catheter was attached to a motorized pullback apparatus and a dedicated
ultrasound scanner (Clearview, Boston Scientific Scimed, Inc). The operator
activated a motor drive that progressively withdrew the IVUS transducer at
a speed of 0.5 mm per second. During the pullback, IVUS images were obtained
at 30 frames per second and recorded on Super-VHS videotape (Figure 1). At follow-up, the operator placed the IVUS catheter in
the same vessel originally imaged, positioned the transducer just distal to
the original fiduciary branch, and initiated a motorized pullback. This procedure
ensured that the identical segment was analyzed at baseline and follow-up.

Figure 1. Method of Intravascular Ultrasound
(IVUS) Interrogation

A motorized pullback of the IVUS imaging catheter is performed beginning
at a distal fiduciary branch (site C) and ending at a proximal branch (site
A). The pathway of the catheter is illustrated in the angiogram in the top
panel. The analysis technician locates the distal branch in the IVUS image,
C, and obtains cross-sections every 0.5 mm until a proximal branch is reached,
A. An intermediate cross-section is illustrated in B.

IVUS Core Laboratory Analysis. Videotapes containing
the IVUS pullbacks were analyzed in the core laboratory by a single operator
(T.T.) blinded to all patient characteristics. The methods for analysis have
been previously described.5 Briefly, the operator
digitized the videotape, reviewed the pullback, and selected the origin of
the most distal side-branch as the beginning point for analysis (Figure 1). Subsequently, every 30th image
was selected for analysis, representing a series of cross-sections spaced
exactly 0.5 mm apart. The final analyzed cross-section was the most proximal
image in the sequence prior to appearance of the left main coronary artery
or right coronary ostium (proximal fiduciary site). In this fashion, a series
of slices were defined at 0.5-mm intervals over a pullback length of 30 mm
to 80 mm (Figure 1). The procedure
was repeated for the follow-up examination using identical landmarks to ensure
that the identical segment was analyzed at both time points.

Direct IVUS Measurements. Intravascular ultrasound
measurements were performed in accordance with the standards of the American
College of Cardiology and European Society of Cardiology.8 Using
National Institutes of Health Image 1.62 (NIH public domain software), the
operator performed a calibration procedure by measuring 1-mm grid marks encoded
in the IVUS image by the scanner. For each cross-section, the operator performed
manual planimetry to trace the leading edges of the luminal and external elastic
membrane borders (Figure 2). The
maximum atheroma thicknesses were also directly measured. The accuracy and
reproducibility of these methods have been previously reported, demonstrating
that, after calibration, mean IVUS cross sectional area measurements were
within 0.5% of actual dimensions for precision-drilled Lucite phantoms ranging
in area from 3.24 mm2 to 27.99 mm2.9 The
variability of measurements by multiple observers demonstrated an SD of 2.9%.

Figure 2. Method of Intravascular Ultrasound
(IVUS) Measurement

The cross-section for analysis is identified, A; the external elastic
membrane (EEM), B, and the lumen area are planimetered, C. Subtraction of
the cross-sectional area of the lumen from the area of the EEM allows calculation
of the atheroma area, D. The maximal atheroma thickness is directly measured,
D.

Derived IVUS Measurements. Atheroma area was
calculated as external elastic membrane (EEM) area minus luminal area. Since
image cross-sections were obtained at 0.5-mm intervals, the total atheroma
volume could be calculated using the Simpson rule as mean atheroma area multiplied
by pullback length in millimeters. The percent atheroma volume was computed
as:

Quantitative Angiography. Analysis of coronary
angiography was performed in a core laboratory at the Cleveland Clinic Foundation
using standardized methods designed to reduce measurement variability. Comparison
of the diameter of the angiographic catheter tip with its known dimension
was used to calibrate the system. The angiographic end point was the change
in the mean coronary luminal diameter from baseline to follow-up.

Statistical Methods

In the protocol, the assumptions used for power calculations required
randomization of 60 patients in a 1:2:2 ratio to placebo and to the low- and
high-dose groups, respectively. The estimated reductions in percent atheroma
volume were −2% in the low-dose group, −4% in the high-dose group,
and −3% for the combined treatment cohort. Assuming an 80% completion
rate, the study would provide 75% power to detect a −3% change in percent
atheroma volume in combined treatment groups (assumed SD of 7%). Categorical
variables are described using frequencies while continuous variables are reported
as mean (SD) values. For the efficacy analyses, the Wilcoxon signed rank test
was performed, using SAS Version 8.02. The 95% confidence intervals (CIs)
were calculated based on the Wilcoxon signed rank test using the method described
by Conover.10

RESULTS

Patient Population

Between November 2001 and March 2003, 123 patients were screened for
inclusion in the study and 57 patients were randomly assigned. A total of
47 patients completed the protocol, 11 in the placebo and 21 in the low-dose
and 15 in the high-ETC-216 groups. Of the 10 patients not completing the study,
2 were withdrawn for an adverse event, 3 withdrew consent, and 5 had IVUS
studies that were not analyzable (the quality of the baseline IVUS study was
considered inadequate by the core laboratory after the patient had been administered
the drug; Figure 3). The demographic,
physical examination, and laboratory characteristics of participants are summarized
in Table 1.

Figure 3. Disposition of Patients in the Trial

ETC-216 indicates apolipoprotein A-I Milano/phospholipid; IVUS, intravascular
ultrasound. *These 4 patients received a single dose of study drug or placebo
but were withdrawn after their baseline IVUS examination was deemed unacceptable
by the core laboratory.

IVUS Baseline Findings

A total of 4016 IVUS cross-sections were analyzed by the core laboratory
at both time points and are included in the analyses. The baseline IVUS findings
are summarized in Table 1. The
mean pullback length was 49.4 mm, containing an average of 86.5 analyzable
cross-sections per patient.

Analyses

Primary Efficacy. The primary prespecified
end point was the change in percent atheroma volume (end of study minus baseline)
in the combined ETC-216 treatment group. This group included all patients
who were randomized in whom an evaluable final IVUS was obtained. A positive
result was defined as a negative change in percent atheroma volume with CIs
not including zero. In the combined treatment group (patients who received
either the low- or high-dose ETC-216), the change in mean (SD) percent atheroma
volume was −1.06% (3.17%). The median was −0.81% (95% CI, −1.53%
to −0.34%; P = .02 compared with baseline; Table 2). For the placebo group, the mean
(SD) change was 0.14% (3.09%). The median was 0.03% (95% CI, −1.11%
to 1.43%; P = .97 compared with baseline).

Subsegmental. To determine the interaction
between observed drug effects and disease severity, the protocol prespecified
analysis of the most severely and least severely diseased 10-mm-long subsegments.
For the combined treatment cohort, the effect of ETC-216 was predominantly
observed as regression of disease in the most severely diseased 10-mm subsegment
(P<.001; Table
5) In the least severely diseased subsegment, no treatment effect
was observed (P = .49, data available on request
from the author).

Exploratory Analyses. Because of the small
size of the trial and numerically greater plaque burden in the actively treated
groups, a post hoc sensitivity analysis was performed using several additional
methods for evaluating efficacy. These included unpaired comparisons of the
combined treatment arm to placebo for the primary and secondary efficacy parameters
adjusting for baseline values (Table 2, Table 3, Table 4, and Table 5).
An additional analysis was performed in which the patients who were randomly
assigned but did not complete the trial were included in the efficacy analyses
and assumed to have no change in plaque burden. Using this alternative analysis
(imputing patients who did not complete the study), the resulting P value for the primary and secondary efficacy parameters were unchanged.

Adverse Events

Adverse events are noted in Table
6. Minor gastrointestinal adverse effects, such as nausea, occurred
in all 3 groups. One patient in the high-dose group developed an elevated
aspartate aminotransferase on a single occasion (>3 × the upper limit
of normal), accompanied by nausea, vomiting, and cholelithiasis and was withdrawn
from the study for an adverse event. Another patient in the high-dose group
experienced a reaction consisting of chills, nausea, diaphoresis, rigors,
vomiting, and mild rash during infusion, deemed possibly drug related and
was withdrawn from the study for an adverse event.

COMMENT

Although epidemiological studies have demonstrated that HDL-C levels
are inversely correlated with atherosclerotic clinical events, the value of
raising HDL-C as a therapeutic target remains uncertain.11 Currently
available drugs have only a modest effect on HDL-C and possess other pharmacological
effects that confound efforts to determine whether observed benefits are related
to alterations in HDL-C levels. Administration of an exogenously produced
HDL mimetic offers the opportunity to explore an entirely new approach to
atherosclerosis treatment. We used a unique form of synthetic HDL, an ApoA-I
Milano/phospholipid complex, because the naturally occurring carriers of ApoA-I
Milano are protected from vascular disease. In this initial study, this new
strategy had a favorable effect on atherosclerotic disease burden despite
a short duration of treatment. An example of regression in a patient treated
with ETC-216 is illustrated in Figure 4.

Figure 4. Example of Atheroma Regression in
a Patient Who Received High-Dose ETC-216

The atheroma area decreased from 8.1 to 5.35 mm2 with
virtually no change in the lumen area. EEM indicates external elastic membrane.

The statistical approach used to assess regression in this study merits
additional comment. In this type of analysis, each patient serves as his or
her own control, with paired comparisons performed between the baseline and
end-of-study measures of atheroma burden. Because this was a pilot study,
the study did not have sufficient power to test the hypothesis that significant
differences between groups would be identified. However, the protocol did
specify analysis of the placebo-treated patients as a separate group to provide
insight into any systematic observer bias in measurement of atheroma burden.
In addition, an intent-to-treat analysis of all patients analyzed as randomized
was not preplanned. However, the results of the study did not change significantly
if no change in atheroma burden was assumed for all patients who withdrew
before study completion. Based on the findings of all analyses, we believe
that the current study provides compelling evidence of atherosclerosis regression
following short-term treatment with the exogenous HDL mimetic ETC-216, but
these results should be confirmed in a larger, long-term study with clinical
end points.

The rapidity and magnitude of the changes in atherosclerotic disease
burden observed in the current study have not been previously observed. Brown
et al12 administered the combination of simvastatin
and niacin and reported a −0.4% change in angiographic percent stenosis
after 3 years of treatment. The regression observed in our study was substantially
larger (−1% for percent atheroma volume and −4.2% for total atheroma
volume) and occurred after only 5 weeks of treatment. Several phenomena may
account for these observations. Therapy with an HDL mimetic may achieve benefit
more quickly and produce a greater extent of regression than conventional
lipid-lowering treatment. Alternatively, IVUS measures changes occurring within
the vessel wall, not just the lumen. A substantial reduction in atherosclerotic
burden may occur in the absence of changes in luminal measurements (Figure 4).

The mechanisms of action of ApoA-1 Milano/phospholipid that result in
regression of atherosclerosis are unknown but presumably are related to an
increase in reverse cholesterol transport from atheromatous lesions to the
serum with subsequent modification and removal by the liver. The cysteine
substitution for arginine at position 173 in the ApoA-1 Milano variant allows
dimerization, forming large HDL particles that may be particularly active
in reverse cholesterol transport. In vitro experiments have demonstrated increased
cholesterol efflux from cholesterol-loaded hepatoma cells incubated with serum
from ApoA-1 Milano carriers or from transgenic mice.13 Because
no previous human studies exist from which to determine the optimal dose of
an exogenous HDL mimetic, we tested 2 different doses of ETC-216, 15 mg/kg
and 45 mg/kg. For the primary and secondary efficacy analyses, there was no
evidence of a greater extent of regression for the higher dose. These data
suggest that ETC-216 is capable of enhancing reverse cholesterol transport
at both dosage levels.

These data provide intriguing insights into the pathophysiology of coronary
atherosclerosis. Previously, this disease was regarded as relatively static,
characterized by steady, gradual progression. Trials using angiography and
carotid ultrasound to measure atherosclerosis progression typically treated
patients for 2 to 3 years.12,14- 16 The
rapid regression observed in our study provides evidence of a more dynamic
process. Treatment strategies designed to enhance reverse cholesterol transport
may work quickly if sufficiently efficacious. Accordingly, we believe that
therapies designed to affect HDL-C represent an important emerging therapeutic
target. These findings suggest the potential for a novel strategy for management
of the patients with ACS. If these results are confirmed, administration of
an agent to stimulate reverse-cholesterol transport could be used in the first
few weeks or months following an acute event. After a period of intense treatment,
ongoing therapy with conventional lipid modulating agents can provide long-term
clinical benefits.17

The application of IVUS in studies of atherosclerosis regression or
progression represents an important emerging trend in research. Intravascular
ultrasound is particularly well suited for this application. Current devices
operate at very high frequencies, 30 MHz or higher, and offer an axial resolution
of less than 150 µm and a temporal resolution of 33 milliseconds.4,7 Unlike angiography, IVUS depicts a
360° image of the vessel wall rather than a 2-dimensional projection of
the lumen.18 Precise motorized pullback enables
reconstruction of the atheroma burden within long segments of the coronary
artery. These properties permit shorter duration studies in smaller numbers
of patients than previously possible from conventional angiographic methods.
Other recent IVUS studies have demonstrated differences in anti-atherosclerotic
treatment strategies with relatively small sample sizes.19,20

This study has several limitations, particularly the small sample size,
which limits interpretation of both safety and efficacy data. However, the
observation of a large treatment effect and statistically consistent results
for the primary and secondary end points is encouraging. Nonetheless, the
current result is a proof-of-concept study that must be explored in larger
trials. Due to the absence of published data linking IVUS changes in plaque
volume to morbidity and mortality, the clinical relevance of changes in disease
burden remain uncertain. However, the relationship between coronary disease
progression rates and clinical events has been previously established in trials
using angiographic end points.21 Accordingly,
we think it is likely that a rapid and large reduction in disease burden will
improve long-term clinical outcome. Nevertheless, demonstration of an effect
on major clinical events is necessary to confirm the clinical utility of this
approach to the treatment of coronary atherosclerosis.

Despite these limitations, several conclusions from this study are warranted.
This initial trial of an exogenously produced HDL mimetic demonstrated significant
evidence of rapid regression of atherosclerosis. The potential utility of
the new approach must be fully explored in a larger patient population with
longer follow-up, assessing a variety of clinical end points, including morbidity
and mortality.

Expert Panel on Detection, Evaluation, and Treatment of High Blood
Cholesterol in Adults. Executive summary of the third report of the National Cholesterol Education
Program (NCEP) expert panel on Detection, Evaluation, and Treatment of High
Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA.2001;285:2486-2497.PubMed

Figures

Figure 1. Method of Intravascular Ultrasound
(IVUS) Interrogation

A motorized pullback of the IVUS imaging catheter is performed beginning
at a distal fiduciary branch (site C) and ending at a proximal branch (site
A). The pathway of the catheter is illustrated in the angiogram in the top
panel. The analysis technician locates the distal branch in the IVUS image,
C, and obtains cross-sections every 0.5 mm until a proximal branch is reached,
A. An intermediate cross-section is illustrated in B.

Figure 2. Method of Intravascular Ultrasound
(IVUS) Measurement

The cross-section for analysis is identified, A; the external elastic
membrane (EEM), B, and the lumen area are planimetered, C. Subtraction of
the cross-sectional area of the lumen from the area of the EEM allows calculation
of the atheroma area, D. The maximal atheroma thickness is directly measured,
D.

Figure 3. Disposition of Patients in the Trial

ETC-216 indicates apolipoprotein A-I Milano/phospholipid; IVUS, intravascular
ultrasound. *These 4 patients received a single dose of study drug or placebo
but were withdrawn after their baseline IVUS examination was deemed unacceptable
by the core laboratory.

Figure 4. Example of Atheroma Regression in
a Patient Who Received High-Dose ETC-216

Expert Panel on Detection, Evaluation, and Treatment of High Blood
Cholesterol in Adults. Executive summary of the third report of the National Cholesterol Education
Program (NCEP) expert panel on Detection, Evaluation, and Treatment of High
Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA.2001;285:2486-2497.PubMed

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